A critical feature of the life cycle the human immunodeficiency virus (HIV-1) that causes Acquired Immunodeficiency Syndrome (AIDS) is its ability to generate diversity. HIV-1 has exceptionally high mutation rates within certain portions of its genome, permitting rapid evolution of new forms of the virus that are able to evade the host's immune response. In order to determine if errors committed by the viral reverse transcriptase could account for diversity in vivo, we had previously examined the accuracy of HIV-1 reverse transcriptase RT using in vitro fidelity assays and found this enzyme to be exceptionally error-prone. Sequence analysis of mutants resulting from in vitro synthesis demonstrates that the enzyme has unusual error specificity. Base substitution and one-base frameshift mutational hotspots are observed. The specificity and position of errors suggest that most mutational hot spots result from template-primer slippage. Using site-directed mutagenesis to alter the template DNA sequence for subsequent use in fidelity assays, we have obtained strong evidence that the frameshifts are indeed due to misalignment. Using a steady-state enzyme kinetic analysis of an exceptional base-substitution hot spot, we have also established equally strong support for a model for base substitutions generated by transient misalignment. Processivity analysis for the enzyme on the M13mp2 DNA template reveals strong termination at specific sites. Termination sites within homopolymer sequences correlate with frameshift mutational hot spots. Since these results suggest that the formation and/or utilization of misaligned template-primers is increased during the dissociation-reinitiation phase of the reaction, we are attempting to further examine this stage of the reaction. Our future work will continue to focus on elucidating the mechanisms responsible for the error-proneness of HIV-1 RT. These studies may provide insights into the interaction of the enzyme's active site with its substrates and may be useful in designing RT-targeted drugs.